Correction: Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

[This corrects the article DOI: 10.1371/journal.pgen.1005792.].

Differential expression patterns of purinergic ectoenzymes and the antioxidative role of IL-6 in hospitalized COVID-19 patient recovery.

Introduction: We have acquired significant knowledge regarding the pathogenesis of severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). However, the underlying mechanisms responsible for disease recovery still need to be fully understood. Methods: To gain insights into critical immune markers involved in COVID-19 etiopathogenesis, we studied the evolution of the immune profile of peripheral blood samples from patients who had recovered from COVID-19 and compared them to subjects with severe acute respiratory illness but negative for SARS-CoV-2 detection (controls). In addition, linear and clustered correlations between different parameters were determined. Results: The data obtained revealed a significant reduction in the frequency of inflammatory monocytes (CD14+CD16+) at hospital discharge vs. admission. Remarkably, nitric oxide (NO) production by the monocyte compartment was significantly reduced at discharge. Furthermore, interleukin (IL)-6 plasma levels were negatively correlated with the frequency of NO+CD14+CD16+ monocytes at hospital admission. However, at the time of hospital release, circulating IL-6 directly correlated with the NO production rate by monocytes. In line with these observations, we found that concomitant with NO diminution, the level of nitrotyrosine (NT) on CD8 T-cells significantly diminished at the time of hospital release. Considering that purinergic signaling constitutes another regulatory system, we analyzed the kinetics of CD39 and CD73 ectoenzyme expression in CD8 T-cells. We found that the frequency of CD39+CD8+ T-cells significantly diminished while the percentage of CD73+ cells increased at hospital discharge. In vitro, IL-6 stimulation of PBMCs from COVID-19 patients diminished the NT levels on CD8 T-cells. A clear differential expression pattern of CD39 and CD73 was observed in the NT+ vs. NT-CD8+ T-cell populations. Discussion: The results suggest that early after infection, IL-6 controls the production of NO, which regulates the levels of NT on CD8 T-cells modifying their effector functions. Intriguingly, in this cytotoxic cell population, the expression of purinergic ectoenzymes is tightly associated with the presence of nitrated surface molecules. Overall, the data obtained contribute to a better understanding of pathogenic mechanisms associated with COVID-19 outcomes.

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells.

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2 deficiency.

BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.

SARS-CoV-2 detection in multi-sample pools in a real pandemic scenario: A screening strategy of choice for active surveillance.

BACKGROUND: The current COVID-19 pandemic has overloaded the diagnostic capacity of laboratories by the gold standard method rRT-PCR. This disease has a high spread rate and almost a quarter of infected individuals never develop symptoms. In this scenario, active surveillance is crucial to stop the virus propagation. METHODS: Between July 2020 and April 2021, 11,580 oropharyngeal swab samples collected in closed and semi-closed institutions were processed for SARS-CoV-2 detection in pools, implementing this strategy for the first time in Córdoba, Argentina. Five-sample pools were constituted before nucleic acid extraction and amplification by rRT-PCR. Comparative analysis of cycle threshold (Ct) values from positive pools and individual samples along with a cost-benefit report of the whole performance of the results was performed. RESULTS: From 2,314 5-sample pools tested, 158 were classified as positive (6.8%), 2,024 as negative (87.5%), and 132 were categorized as indeterminate (5.7%). The Ct value shift due to sample dilution showed an increase in Ct of 2.6±1.53 cycles for N gene and 2.6±1.78 for ORF1ab gene. Overall, 290 pools were disassembled and 1,450 swabs were analyzed individually. This strategy allowed correctly identifying 99.8% of the samples as positive (7.6%) or negative (92.2%), avoiding the execution of 7,806 rRT-PCR reactions which represents a cost saving of 67.5%. CONCLUSION: This study demonstrates the feasibility of pooling samples to increase the number of tests performed, helping to maximize molecular diagnostic resources and reducing the work overload of specialized personnel during active surveillance of the COVID-19 pandemic.

Synthetic Lethal Activity of Benzophenanthridine Alkaloids From Zanthoxylum coco Against BRCA1-Deficient Cancer Cells.

Several plants from South America show strong antitumoral properties based on anti-proliferative and/or pro-apoptotic activities. In this work we aimed to identify selective cytotoxic compounds that target BRCA1-deficient cancer cells by Synthetic Lethality (SL) induction. Using a high-throughput screening technology developed in our laboratory, we analyzed a collection of extracts from 46 native plant species from Argentina using a wide dose-response scheme. A highly selective SL-induction capacity was found in an alkaloidal extract from Zanthoxylum coco (Fam. Rutaceae). Bio-guided fractionation coupled to HPLC led to the identification of active benzophenanthridine alkaloids. The most potent SL activity was found with the compound oxynitidine, which showed a remarkably low relative abundance in the active fractions. Further validation experiments were performed using the commercially available and closely related analog nitidine, which showed SL-induction activity against various BRCA1-deficient cell lines with different genetic backgrounds, even in the nanomolar range. Exploration of the underlying mechanism of action using BRCA1-KO cells revealed AKT and topoisomerases as the potential targets responsible of nitidine-triggered SL-induction. Taken together, our findings expose an unforeseen therapeutic activity of alkaloids from Zanthoxylum-spp. that position them as novel lead molecules for drug discovery.

Synthesis and structure-activity relationships of novel abietane diterpenoids with activity against Staphylococcus aureus.

Aim: To find alternative compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), novel derivatives from dehydroabietic acid were synthesized. Methods & results: Compound 12 was the most effective against 15 MRSA and 11 MSSA with minimum inhibitory concentration values ranging from 3.9 to 15.6 µg/ml. Although less active than 12, compound 11, followed by 25 and 13, also exhibited anti-staphylococcal activity. Additional studies showed that compound 12 is devoid of toxic effect on non-target cells. A structure-activity relationship study revealed that an oxime at C-13 together with a hydroxyl at C-12 could play a key role in the activity. Conclusion: These structures, in particular compound 12, could arise as templates for the development of agents against MRSA and MSSA.

Krüppel-Like Factor 6 Is Required for Oxidative and Oncogene-Induced Cellular Senescence.

Krüppel-like factor 6 (KLF6) is a transcription factor involved in the regulation of several cellular processes. Regarding its role in tumorigenesis, KLF6 is considered a tumor suppressor. Numerous reports demonstrate its frequent genomic loss or down-regulation, implying a functional inactivation in a broad range of human cancers. Previous work from our laboratory showed that the down-regulation of KLF6 expression in normal fibroblasts leads to cellular transformation, while its ectopic expression interferes with the oncogenic transformation triggered by activated Ras through a cell cycle arrest. We hypothesize that the growth suppressor activity of KLF6 may involve the induction of cellular senescence thereby helping to prevent the proliferation of cells at risk of neoplastic transformation. Here, we explored the association of KLF6 up-regulation in two different cellular senescence scenarios. We found that KLF6 silencing bypasses both oxidative and oncogene-induced senescence. In this context, KLF6 expression per se was capable to trigger cellular senescence in both normal and tumoral contexts. As such, the findings presented in this report provide insights into a potential mechanism by which KLF6 may play a suppressing role of uncontrolled or damaged cell proliferation.

PKCα Modulates Epithelial-to-Mesenchymal Transition and Invasiveness of Breast Cancer Cells Through ZEB1.

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction.

PURPOSE: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. EXPERIMENTAL DESIGN: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. RESULTS: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. CONCLUSIONS: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress.

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.

Downregulation of adaptor protein MyD88 compromises the angiogenic potential of B16 murine melanoma.

The mechanisms that link inflammatory responses to cancer development remain a subject of intense investigation, emphasizing the need to better understand the cellular and molecular pathways that create a tumor promoting microenvironment. The myeloid differentiation primary response protein MyD88 acts as a main adaptor molecule for the signaling cascades initiated from Toll-like receptors (TLRs) and the interleukin 1 receptor (IL-1R). MyD88 has been shown to contribute to tumorigenesis in many inflammation-associated cancer models. In this study, we sought to better define the role of MyD88 in neoplastic cells using a murine melanoma model. Herein, we have demonstrated that MyD88 expression is required to maintain the angiogenic switch that supports B16 melanoma growth. By knocking down MyD88 we reduced TLR-mediated NF-κB activation with no evident effects over cell proliferation and survival. In addition, MyD88 downregulation was associated with a decrease of HIF1α levels and its target gene VEGF, in correlation with an impaired capability to induce capillary sprouting and tube formation of endothelial cells. Melanomas developed from cells lacking MyD88 showed an enhanced secretion of chemoattractant ligands such as CCL2, CXCL10 and CXCL1 and have an improved infiltration of macrophages to the tumor site. Our results imply that cell-autonomous signaling through MyD88 is required to sustain tumor growth and underscore its function as an important positive modulator of tumor angiogenesis.

P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach.

This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF-CEM, K562, A549 and HCT116 cells, with a highly selective effect on the latter, with an IC50 value of 0.2 µM. Based on this finding, HCT116 cells were selected to study the mechanism of action of 1. Cell cycle analysis revealed that 1 induced sustained arrest in the S-phase, which was followed by the triggering of apoptotic cell death and reduced clonogenic capacity. This cytotoxicity was seen to be preceded by the upregulation of the tumor suppressor p53 and its target effector p21. In addition, it was found that p53 expression was required for efficient cell death induction, and thus that the toxicity of 1 relies mainly on p53-dependent mechanisms. Taken together, these findings position 1 as a potent antitumor agent, with potential for the development of novel chemotherapeutic drugs based on the induction of S-phase arrest.

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na(+)/I(-) Symporter Expression.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis.

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2',4'-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4'-dihydroxy-3'-methoxyflavanone (5). Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 µg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1-5 was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6-9.9 µM) and a lower effect against CML cells (IC50 = 27.5-30.0 µM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

Krüppel-like factor 6 interferes with cellular transformation induced by the H-ras oncogene.

KLF6 is a member of the Krüppel-like factor family of transcription factors, with diverse roles in the regulation of cell physiology, including proliferation, signal transduction, and apoptosis. Mutations or down-regulation of KLF6 have been described in several human cancers. In this work, we found that KLF6-knockdown resulted in the formation of transformed foci and allowed the spontaneous conversion of NIH3T3 cells to a tumorigenic state. We further assessed the role of KLF6 in the context of oncogenic Ras. We showed that KLF6 was up-regulated by H-Ras(G12V) expression in a Jun N-terminal kinase (JNK)-dependent manner, correlated with enhanced klf6 promoter activity. We found that ectopic KLF6 expression induced a G1-phase cell cycle arrest, thereby decreasing the cell proliferation rate. In addition, constitutive KLF6 expression impaired H-Ras(G12V)-mediated loss of density-dependent growth inhibition and anchorage-independent growth. Moreover, growth of H-Ras(G12V)-driven tumors was reduced in mice challenged with cells stably expressing KLF6. KLF6 expression correlated with the up-regulation of p21, whereas neither p53 induction nor apoptotic cell death was detected. Further, p21 knockdown impaired KLF6-induced cell cycle arrest. These findings provide novel evidence highlighting KLF6 function in response to malignant transformation, suggesting the relevance of KLF6 in controlling cell proliferation and hindering tumorigenesis.

Biology of Krüppel-like factor 6 transcriptional regulator in cell life and death.

An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.

GH3B6 pituitary tumor cell proliferation is mediated by PKCalpha and PKCepsilon via ERK 1/2-dependent pathway.

BACKGROUND: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. METHODS: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. RESULTS: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. CONCLUSION: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.